Infectious Moloney murine leukemia virus (M-MuLV) and HIV are non-infectious when the genome is modified by either of two point mutations that change codons for two conserved amino acids in the C-terminal region of reverse transcriptase (RT). We have demonstrated in partially-purified cloned RTs from each of these viruses that polymerase is essentially as active as wild-type RT; however, the RNase H function is severely restricted. Defective M-MuLV particles produced by transformed NIH 3T3 cells were used to infect normal 3T3 cells to determine the kinetics of minus strand DNA synthesis by PCR analysis. Infectious and mutant virus produced (-) strong stop DNA and the "jump" within 15 min after infection, but minus strand DNA did not extend beyond U3. These results confirm in an in vivo system that polymerase function in the mutant virus is adequate, whereas inadequate RNase H function is probably the cause of the defect. The HIV RT assay performed on infected culture supernatant fluids is used as an indicator for the presence of HIV. This assay is not directly proportional to virus concentration and it lacks the sensitivity of the p24 ELISA assay. We have analyzed the requirements for RT activity and have developed an assay that now responds proportionally to enzyme concentration, and the assay is almost as sensitive as p24 detection (a factor of 2 - 3).